LincRNA‐EPS impairs host antiviral immunity by antagonizing viral RNA–PKR interaction

Abstract LincRNA‐EPS is an important regulator in inflammation. However, the role of lincRNA‐EPS in the host response against viral infection is unexplored. Here, we show that lincRNA‐EPS is downregulated in macrophages infected with different viruses including VSV, SeV, and HSV‐1. Overexpression of lincRNA‐EPS facilitates viral infection, while deficiency of lincRNA‐EPS protects the host against viral infection in vitro and in vivo. LincRNA‐EPS −/− macrophages show elevated expression of antiviral interferon‐stimulated genes (ISGs) such as Mx1, Oas2, and Ifit2 at both basal and inducible levels. However, IFN‐β, the key upstream inducer of these ISGs, is downregulated in lincRNA‐EPS −/− macrophages compared with control cells. RNA pulldown and mass spectrometry results indicate that lincRNA‐EPS binds to PKR and antagonizes the viral RNA–PKR interaction. PKR activates STAT1 and induces antiviral ISGs independent of IFN‐I induction. LincRNA‐EPS inhibits PKR‐STAT1‐ISGs signaling and thus facilitates viral infection. Our study outlines an alternative antiviral pathway, with downregulation of lincRNA‐EPS promoting the induction of PKR‐STAT1‐dependent ISGs, and reveals a potential therapeutic target for viral infectious diseases.


8th Oct 2021 1st Editorial Decision
Dear Dr. Ma, Thank you for the submission of your manuscript to EMBO reports. We have now received the full set of referee reports as well as referee cross-comments that are pasted below.
As you will see, the referees acknowledge that the findings are potentially interesting. However, they also point out that significant revisions will be required before the study can be considered for publication here. The referees note that the link between lincRNA-EPs and PKR is weak, that the role of STAT1 should be examined, that it should be clarified which genes are PKR-STAT1 regulated and which genes are hnRNPL-EPS controlled, that primary BMDM from lincRNA-EPS deficient mice should be used, and that an endogenous interaction of lincRNA-EPS with PKR should be shown. We agree that all these are important points, thus all referee concerns should be addressed. If you like, we can also talk about the revision requirements in a video chat.
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As part of the EMBO publication's Transparent Editorial Process, EMBO reports publishes online a Review Process File (RPF) to accompany accepted manuscripts. This File will be published in conjunction with your paper and will include the referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript.
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Main points: 1-The observation that EPS is rapidly decreased upon infection with VSV is not very surprising given that this has previously been observed with pIC and SeV (Atianand et al 2016). That specific Atianand paper also showed that EPS deletion indeed lead to basal upregulation of several key ISGs (indeed highlighted in Fig 5E of this paper), with several having direct antiviral effects (such as RSAD2, MX1, IFIT1). To this reviewer it is hard to see what the first 5 figures of the current paper really add to this previous knowledge aside from the in vivo experiment (we already knew that EPS deficiency was boosting select ISG expression, so it seems incremental to see that there is less viral replication with the viruses shown). There are several issues in claiming that IFNb is not regulated (since basally it is actually up in EPS KO) when using a viral infection. The data from figure 4E should be complemented by looking at IFNb induction with pIC and pdAdT; similarly the WB fig5H should be repeated with pIC as an inducer rather than the virus (since the viral replication is altered due to basal ISG expression).
2-The link with PKR is tenuous. This reviewer notes that there is an estimated 11 copies of EPS RNA per cell (based on the Cell paper cited above), and it is hard to imagine that these molecules would be naturally interacting with PKR when they are principally bound to nuclear chromatin (this needs to be discussed). Critically, most of the data linking PKR is based on RNAi with a single siRNA. Given the off-target effects of RNAi, it is essential to have at least two independent siRNAs targeted to the same target and showing the same phenotype to support any conclusions made. The theory of the authors does not explain how in the absence of infection, the ISG (STAT1 targets) they picked would be induced by the PKR-STAT1 axis (noting that STAT1-P is entirely absent in mock cells Fig 5H). Critically, if the PKR-STAT1 axis is really involved, then RNAi of STAT1 should also decrease the basal expression of selected ISGs basally engaged in EPS cells.
3-The authors entirely omit the concept presented in the Atianand paper that EPS Maintains a Repressed Chromatin State at selected IRGs (including key antiviral genes) -which directly explains why some antiviral genes are basally up-regulated in EPS KO cells. Which genes would be PKR-STAT1 regulated and which genes would be hnRNPL-EPS controlled??? Since many antiviral genes such as RSAD2 and IFIT1 were found to be regulated by the hnRNPL-EPS axis it is hard to distinguish the antiviral effects of the proposed PKR-STAT1 axis (but this is an essential thing to perform experimentally to substantiate the overall point of the paper). Referee #2: lincRNA-EPS has been previously characterized as an immunoregulatory lncRNA that restrains inflammatory responses, although its role in antiviral immunity is unknown. Here the demonstrate that lincRNA-EPS as a negative regulator of host antiviral responses. They report that lincRNA-EPS is downregulated during RNA virus infection, to allow an adequate anti-viral response. The authors demonstrate that LincRNA-EPS binds PKR to block its interaction with viral RNA. Blockade of the PKRviral RNA interaction impairs activation of STAT1 and ISG expression rendering mice more susceptible to RNA virus infection. The results are interesting and add to our knowledge of lncEPS and lncRNA dependent regulation of antiviral immunity more broadly.
There is an overreliance on immortalized cells and overexpression systems. The paper would be strengthened if some of the key observaions could be validated in primary cells and an endogenous interaction of lincRNA-EPS and PKR examined.
Major comments: 1. Figure 1J should include an analysis of absolute copy number of lincRNA-EPS. RNA FISH analysis could also be highly informative here. 2. Throughout the figures are 1 experiment. Instead they should include the mean of all independent experiments performed. 3. It's unclear why immortalized cells were used in Figure 3-5 rather than primary BMDM from lincRNA-EPS deficient mice and WT littermate controls. The main findings of these figures should be repeated using primary BMDMs. 4. The decrease in viral titer in the serum of lincRNA-EPS mice ( Figure 3J) is very modest when compared to WT and is not consistent with the robust phenotype observed in Figure 3G. It would be beneficial to assess the viral titer in the liver and lungs after infection. 5. Figure 4 should include protein levels of at least some representative ISGs. 6. The authors should include a full list of lincRNA-EPS interacting proteins identified by mass spectrometry. Additional controls such as the use of biotinylated lincRNA-EPS probes in lincRNA-EPS deficient BMDMs would further strengthen this part of the study. 7. Figure 7C-G demonstrate upregulated expression of ISGs in mock cells deficient in lincRNA-EPS when compared to WT cells. However, no phospo-STAT1 was detected in lincRNA-EPS KI cells ( Figure 5H). The authors should explain this discrepancy. 8. Figure 7I: a better explanation of the proposed mechanism including subcellular localization should be provided.
Minor comments: 1. Figure 4A: The authors should provide a heatmap demonstrating a full list of genes and pathways detected by the assay. 2. Figure 5F is missing a legend. 3. Figure 6D should include detection of PKR in naïve cells as an additional negative control. 4. Figure 7A: Can the author explain why total STAT1 is downregulated in PKR deficient cells?
Referee #3: In this study, Zhu et al. describe a role of LincRNA-EPS in the negative regulation of STAT1-triggered type I IFN responses. PKR is well-known as a nucleic acids receptor of viral dsRNA. PKR activity needs to be restricted in the resting state to prevent autoimmune activation. The authors claim that lincRNA-EPS binds to PKR and antagonizes the viral RNA-PKR interaction and then inhibits PKR-induced STAT1 activation, resulted in the suppression of downstream ISGs production. Virus infection significantly decreases LincRNA-EPS expression and subsequently removes the restrictions of PKR activation. Therefore, this study help us to understand the self-limiting of the innate immune response. Overall, the findings are novel and interesting. The experiments are well performed, and the manuscript is fluent and generally well organized. However, some concerns mentioned below must be addressed before the manuscript can be considered for publication.
Major comments: 1.As the author describes that PKR is required for phosphorylation and activation of STAT1 under the response to IFN-γ and LPS, but PKR or LincRNA-EPS inhibition greatly changed the total protein level of STAT1 (Fig.5H, Fig.6A and 6B). Activation and phosphorylation of STAT1 should not affect its total protein level, is there any other mechanism for regulation of STAT1? 2.In Fig. 7C, 7E and 7G, WT-siPKR almost blocked VSV infection-induced ISGs expression, but LincRNA-EPS-/--siPKR greatly enhanced ISGs expression compared with WT-siPKR. Therefore PKR knockdown could not change the positive effect of lincRNA-EPS knockout on ISG formation, these results strongly support the existence of other mechanisms that regulate JAK-STAT signaling by LincrNA-EPS. The authors should use IFN-β stimulation instead of VSV infection to investigate whether this phenomenon is caused by viral infection specificity. 3.The H3K4me3 is associated with high transcription activity. However, LincrNA-EPS knockout does not affect the IFN-β induced H3K4me3 of ISGs in the activation state but not the rest state( Fig. 5G and 5H). What are the reasons for the different effect？ 4.The authors claim that lincRNA-EPS regulates STAT1 activation and subsequently controls ISGs expression. However, the study only examined the effects of lincRNA-EPS on virus replication and tissue damage in vivo. Whether lincRNA-EPS could control ISGs expression in tissues (such as lung, spleen or liver) or serum? 5.In Fig. 6H and 6I, lincRNA-EPS overexpression competitively binds virus RNA, which in turn inhibits PKR activation, but more evidence is needed to support this conclusion. Whether lincRNA-EPS knockout or knockdown could promote PKR binds RNA? Whether other dsRNA(such as poly(I: C) have similar phenomena to the VSV-RNA?
Minor comments: 1.The graphic symbols are missing in Fig.5F and 5G. 2.Some description of the background of VSV-N and G is required. 3.In Fig. 3G, the survival percentage of the lincRNA-EPS knockout group did not appear to be calculated from 100%.
Further comments from referee 1: If the authors conducted an RNAseq experiment of EPS-WT and EPS-KO after PKR down-regulation, it may help provide insights into the claimed PKR/EPS-dependent ISG signature -which should only be a portion of the ISGs see in EPS-WT. KO or down-regulation of such PKR-dependent ISGs in the context of EPS-KO cells with infection would be the only direct way to link EPS-PKR targets and antiviral effects.

Dear Dr. Esther Schnapp,
We are submitting our revised manuscript entitled "LincRNA-EPS negatively regulates host antiviral immunity by antagonizing viral RNA interacting with PKR" (EMBOR-2021-53937V1) to EMBO Reports for consideration of publication.
We appreciate all the comments and suggestions from you and all three reviewers. According to the comments and suggestions, we have revised our manuscript by adding new experimental data (Figures 3F, 3I, 3J, 4B, 4D, 5G, 5H, 5I, 6B, 6C, 6I, and Figures EV1D, EV3A, EV4, EV5) and editing our manuscript carefully. The RNA-Seq data analysis (Appendix Figure S1) have been also included.
We have provided point-by-point response as below and indicated the changes (yellow highlighted) in our revised manuscript.
We think that we have addressed all the concerns raised by you and the reviewers.
As a result, the manuscript is indeed significantly improved. Thank you very much for your consideration. We are looking forward to hearing from you.
With my best regards Subject: EMBOR-2021-53937V1 Decision Letter As you will see, the referees acknowledge that the findings are potentially interesting. However, they also point out that significant revisions will be required before the study can be considered for publication here. The referees note that the link between lincRNA-EPS and PKR is weak, that the role of STAT1 should be examined, that it should be clarified which genes are PKR-STAT1 regulated and which genes are hnRNPL-EPS controlled, that primary BMDM from lincRNA-EPS deficient mice should be used, and that an endogenous interaction of lincRNA-EPS with PKR should be shown. We agree that all these are important points, thus all referee concerns should be addressed. If you like, we can also talk about the revision requirements in a video chat. provided. In addition, RNA-Seq analysis was also performed to distinguish the PKR-STAT1 regulated genes and the hnRNPL-EPS controlled genes (Appendix Figure S1).

============================================================= Referee #1:
In this manuscript, Zhu et al. investigate the expression of lincRNA-EPS following viral infection of mouse BMDMs and discovered that it was rapidly down-regulated (Fig 1). Type-I IFN treatment of the cells also promoted this decrease, but the decrease was still seen in Ifnar1 KO cells (albeit modestly decreased) indicating that the effect was partially through another pathway -which the authors propose to be NF-kB using a broad inhibitor. The authors go on to demonstrate that overexpression (12-fold) favors viral infection while CRISPR CAS-9 in RAW cells decreased infection. The authors confirmed these observations in immortalized BMDMS from EPS-KO mice, which were protected against VSV in vivo. Importantly, the mice lacking EPS exhibited less VSV and decreased IFNb levels. RNA-seq signature revealed that several ISGs were increased upon EPS deficiency basally and upon infection (and decreased upon overexpression). Deficiency in EPS lead to basal increased transcriptional activity of MX1 and OAS2, and was associated with increased STAT1 phosphorylation. The authors then show evidence that PKR binds EPS and that RNAi of PKR alleviates some of the antiviral effects of EPS -/-. This leads the authors to conclude that EPS is a key regulator of antiviral responses through its modulation of PKR-driven STAT1. Overall, there are several key issues that need to be addressed to clarify the line of thought of the manuscript and support its conclusions and novelty.

Response:
We appreciate your evaluation and valuable comments for our manuscript.
According to your suggestions, we have revised our manuscript by adding new experimental data ( Figure 5I, EV1D, EV4, EV5, and Appendix Figure S1) and editing our manuscript carefully. Hopefully we have addressed all your concerns.
Point-by-point response to your question has been provided as below.
Main points:

1-The observation that EPS is rapidly decreased upon infection with VSV is not very surprising given that this has previously been observed with pIC and SeV (Atianand et al 2016). That specific Atianand paper also showed that EPS deletion indeed lead to basal upregulation of several key ISGs (indeed highlighted in Fig 5E of this paper), with several having direct antiviral effects (such as RSAD2, MX1, IFIT1). To this reviewer it is hard to see what the first 5 figures of the current paper really add to this previous knowledge aside from the in vivo experiment (we already knew that EPS deficiency was boosting select ISG expression, so it seems incremental to see that there is less viral replication with the viruses shown). There are several issues in claiming that IFNb is not regulated (since basally it is actually up in EPS KO) when using a viral infection. The data from figure 4E should be complemented by looking at IFNb induction with pIC and pdAdT; similarly, the WB fig5H should be repeated with pIC as an inducer rather than the virus (since the viral replication is altered due to basal ISG expression).
Response: Thanks for your comments and suggestion. We admit that the study by  (Fig EV5A), despite not as dramatic as that in VSV infection experiment (Fig 5A and B). The Western blot results shown in Fig 5I also supported the conclusion that knockout of lincRNA-EPS activate stronger IFNAR downstream signaling independent of IFN-. Moreover, although we failed to get the detectable phosphorylation of IRF3 in the polydA:dT-transfected iBMMs, higher induction of pSTAT1, IFIT2, but less pTBK1 in the lincRNA-EPS -/cells were also consistent with the conclusion obtained from the VSV-infected iBMMs (data were shown as below).

2-
The link with PKR is tenuous. This reviewer notes that there are an estimated 11 copies of EPS RNA per cell (based on the Cell paper cited above), and it is hard to imagine that these molecules would be naturally interacting with PKR when they are principally bound to nuclear chromatin (this needs to be discussed). Critically, most of the data linking PKR is based on RNAi with a single siRNA. Given the off-target effects of RNAi, it is essential to have at least two independent siRNAs targeted to the same target and showing the same phenotype to support any conclusions made. The theory of the authors does not explain how in the absence of infection, the ISG (STAT1 targets) they picked would be induced by the PKR-STAT1 axis (noting that STAT1-P is entirely absent in mock cells Fig 5H). Critically, if the PKR-STAT1 axis is really involved, then RNAi of STAT1 should also decrease the basal expression of selected ISGs basally engaged in EPS cells.
Response: Thanks for your comments and insight questions.  (Fig 1J) and there are about 23 copies per cell of lincRNA-EPS in the iBMMs. Therefore, we think it is possible that cytoplasmic lincRNA-EPS is sufficient to interact with PKR, block viral RNA, and thus suppress host antiviral immunity.
2) To eliminate the potential off-target effect of RNAi, we designed two non-overlapping siRNA targeting PKR. Results from both siPKR consistently suggest that upregulation of antiviral ISGs in the lincRNA-EPS -/-iBMMs is required PKR (Figs 7C-H, EV5D-E).
3) Although pSTAT1 is undetectable in the mock WT and lincRNA-EPS -/-iBMMs, we still believe that the PKR-STAT1 signaling maintains basal level of ISG expression. Low level type I IFN and pSTAT1 maintains multiple basal ISG expression such as Mx1 and IFIT2 genes (PMID:30517866). We performed the experiment as you suggested, and found that knockdown of STAT1 also significantly decrease the basal expression of selected ISGs such as Mx1 and Oas2 in the lincRNA-EPS -/-iBMMs (data has been shown as below). It will be more convincing if the lincRNA-EPS -/-STAT1 -/cells are available to verify these RNAi results in future.

3-
The authors entirely omit the concept presented in the Atianand paper that EPS Maintains a Repressed Chromatin State at selected IRGs (including key antiviral genes) -which directly explains why some antiviral genes are basally up-regulated in EPS KO cells. Which genes would be PKR-STAT1 regulated and which genes would be hnRNPL-EPS controlled??? Since many antiviral genes such as RSAD2 and IFIT1 were found to be regulated by the hnRNPL-EPS axis it is hard to distinguish the antiviral effects of the proposed PKR-STAT1 axis (but this is an essential thing to perform experimentally to substantiate the overall point of the paper). Therefore, we think that hnRNPL-EPS axis and PKR-STAT1 axis synergistically control ISGs expression but not work separately.

... what is the biological relevance of a 12-fold increase of EPS RNA?
Response: Thanks for your insight questions. Given that the fold change of lincRNA-EPS transcript during viral infection is between 2 to 20 folds ( Fig 1A) and the variation of lincRNA-EPS expression between macrophages is less than 20 folds ( Fig EV1D), we chose moderate overexpression rather than the high overexpression to investigate the role of lincRNA-EPS during viral infection. Although we can artificially overexpress lincRNA-EPS over 100 folds, moderate upregulation is more reasonable to study the physiological and pathophysiological function of lincRNA-EPS.

2-Fig 5F/G is missing color keys
Response: Thanks for your reminding. We have added the color keys in Fig 5G of

3-Why are you using immortalized BMDMs from EPS -/mice (which end up being similar to your RAW cells) and not primary cells?
Response: Thanks for your insight question. Actually, we have tested the function of lincRNA-EPS during viral infection by using several macrophages including two macrophage cell lines (RAW264.7 and iBMMs) and two primary macrophages (PM and BMDM). RAW264.7 and iBMMs always showed the robust and consistent phenotypes. Moreover, it is much easier for gene overexpression, knockdown, and viral infection in the iBMMs than the primary macrophages. Therefore, we used iBMMs more often than primary macrophages. However, we have confirmed several key experiments by using both PMs (Fig 3F and Fig 4D) and primary BMDMs ( Fig   EV4A) to make our study more physiological relevance. Based on our finding, it is likely that more copies of lincRNA-EPS in the iBMMs makes us easier to studies the function of lincRNA-EPS (Fig EV1D).

4-Fig 6F should be relative to each siRNA (siPKR increased viral load in WT so cannot be 1).
Response: Thanks for your suggestion. The increased viral load in the siPKR groups was shown in Fig 6E. However, in order to indicate the decrease rate of VSV titer in the siNC and siPKR groups, we used the parameter (VSV titer)/(VSV titer) WT . (VSV titer) WT /(VSV titer) WT should be 1 and (VSV titer) ko /(VSV titer) WT should be less than 1. Results shown in Fig 6F could

There is an overreliance on immortalized cells and overexpression systems. The paper would be strengthened if some of the key observations could be validated in primary cells and an endogenous interaction of lincRNA-EPS and PKR examined.
Response: We appreciate your comments and suggestions, which significantly improve the value of our study. As you suggested, we have performed the experiments by using both primary peritoneal macrophages and BMDMs. These Major comments: 1. Figure 1J should include an analysis of absolute copy number of lincRNA-EPS. RNA FISH analysis could also be highly informative here.
Response: Thanks for your suggestion. We have measured and calculated the absolute copy number of lincRNA-EPS transcript in the cell types that we used by RT-qPCR. As shown in Fig EV1D, RAW264.7 and iBMMs express about 63 and 23 copies lincRNA-EPS per cell, respectively, higher than BMDM (~8 copies/cell).
Based on our finding, it is likely that more abundance of lincRNA-EPS in the iBMMs makes us easier to study the function of lincRNA-EPS.

Throughout the figures are 1 experiment. Instead, they should include the mean of all independent experiments performed.
Response: Thanks for reminding us to check the data analyzing and statistics.
Actually, we performed most of experiments with biological triplicates, which should be considered as three independent experiments. In some cases, we used one representative results of three independent experiments. We have included the mean of all independent experiments performed and corrected the description in the figure legends of the revised manuscript. Figure 3 Figure 3J) is very modest when compared to WT and is not consistent with the robust phenotype observed in Figure 3G. It would be beneficial to assess the viral titer in the liver and lungs after infection.

The decrease in viral titer in the serum of lincRNA-EPS mice (
Response: Thanks for your comments which will greatly improve the significance of our study. In the survival rate experiment, we injected lethal dose of VSV intravenously (1×10 8 PFU/g) to cause most of WT mice dead at 24 h post infection.
However, in order to get enough samples, especially the serum sample in the VSV-infected WT mice, we decrease the infection viral titer to sub-lethal dose by intraperitoneally injection, which may partially explain the modest phenotype in the serum viral load when comparing to the survival results. In the revised manuscript, we repeated this key experiment by injecting VSV with titer of 6×10 7 PFU/g intravenously and get more robust phenotype on viral load (left panel of Fig 3I). In addition, the viral titer in the livers and lungs are checked and the results are shown in revised Fig 3I (middle and right panels). Figure 4 should include protein levels of at least some representative ISGs.

5.
Response: Thanks for your suggestions. As shown in the Fig 5H and I, we added the protein bands of antiviral ISG IFIT2 during iBMMs infected with VSV or transfected with polyI:C. Both IFIT2 transcripts and proteins levels are higher induced in lincRNA-EPS -/-iBMMs than WT cells during antiviral immunity.

The authors should include a full list of lincRNA-EPS interacting proteins identified by mass spectrometry. Additional controls such as the use of biotinylated lincRNA-EPS probes in lincRNA-EPS deficient BMDMs would further strengthen this part of the study.
Response: Thanks for your suggestions. We have provided Table EV1 which Table EV1. We also pulled down the target protein PKR from both WT and lincRNA-EPS -/-iBMMs by using biotinylated lincRNA-EPS (revised Fig 6B). Because of the slightly elevated PKR and more dissociated PKR from PKR-lincRNA-EPS complex, much more PKR was pulled down by the biotinylated lincRNA-EPS in the lincRNA-EPS -/-iBMMs than in the WT cells. In addition, biotinylated polyI:C, another potent ligand of PKR, also pulled down more PKR in the lincRNA-EPS -/-iBMMs than in the WT cells. Figure 5H). The authors should explain this discrepancy.

Figure 7C-G demonstrate upregulated expression of ISGs in mock cells deficient in lincRNA-EPS when compared to WT cells. However, no phospo-STAT1 was detected in lincRNA-EPS KI cells (
Response: Thanks for your comments and suggestion. We think that the basal level of phosphorylated STAT1 is difficult to be detected because of the limited sensitivity of pSTAT1 antibody. However, basal pSTAT1 exists since the constitutive expression of low-level type I IFNs. Actually, basal type I IFN or pSTAT1 maintains multiple ISG expression such as Mx1 and IFIT2 genes (PMID:30517866). Figure 7I: a better explanation of the proposed mechanism including subcellular localization should be provided. Minor comments: 1. Figure 4A: The authors should provide a heatmap demonstrating a full list of genes and pathways detected by the assay.

Response
Response: Thanks for your suggestion. We have provided the heatmaps for ISGs ( Fig   4B of the revised manuscript) and the DEGs belong to the top three pathways of GSEA ( Fig 4A and Fig EV3A of the revised manuscript). Figure 5F is missing a legend.

2.
Response: Thanks for your reminding. We have added the legend in the revised Fig   5G. 3. Figure 6D should include detection of PKR in naïve cells as an additional negative control.
Response: Thanks for your suggestion. We have included an additional control (siRNA without transfection reagent, labeled as "Ctrl") in the Fig EV5B of the revised manuscript. In addition, the knockdown efficiency of two siRNAs targeting PKR is clearly shown in the revised Fig EV5B. 4. Figure 7A: Can the author explain why total STAT1 is downregulated in PKR deficient cells?
Response: Thanks for your question. We used two non-overlapping siRNA targeting PKR to eliminate the off-target effect of RNAi, and observed the downregulation of STAT1 in both knockdown cells (Fig EV5B). Overexpression of PKR in MEF cells also upregulated STAT1 expression and STAT1 phosphorylation (Fig EV5C). Given that STAT1 is also an ISG, it is possible that PKR-STAT1 signaling proposed in Fig   7I (left panel) also controls STAT1 itself expression. However, the detail mechanism that explain how PKR regulates STAT1 activation needs to be further studied.
According to our published and unpublished studies, we think that multiple kinases work similar as Jak1 which directly activates STAT1. Point-by-point response to your individual question has been provided as below.
Major comments: 1.As the author describes that PKR is required for phosphorylation and activation of STAT1 under the response to IFN-γ and LPS, but PKR or LincRNA-EPS inhibition greatly changed the total protein level of STAT1 (Fig5H, Fig7A and 7B). Activation and phosphorylation of STAT1 should not affect its total protein level, is there any other mechanism for regulation of STAT1?
Response: Thanks for your question. According to our conclusions, knockout of lincRNA-EPS removes the restrictions of PKR activation, which may maintain basal STAT1 activation and ISG induction via the PKR-STAT1 signaling. Given that STAT1 itself is an ISG (PMID: 19478064), it is possible that PKR-STAT1 signaling proposed in Fig 7I (left panel) also controls STAT1 itself expression. Therefore, it is reasonable to observe elevated and downregulated STAT1 expression in the lincRNA-EPS -/and PKR knockdown iBMMs, respectively. In addition, we have confirmed that overexpression of PKR in MEF cells also upregulates STAT1 expression and this induction is suppressed by lincRNA-EPS (Fig EV5C), which is consistent with the results observed the in the lincRNA-EPS -/and PKR knockdown cells. We don't think the inhibitory function lincRNA-EPS is viral infection specific, since the results based on viral mimics polyI:C shows viral mimics also compete with lincRNA-EPS to interacts with PKR (revised Fig 6B and I). Similar as VSV infection, polyI:C also induced more pSTAT1 and ISG in the lincRNA-EPS -/-iBMMs than in the WT cells (revised Fig 5I).

Response
3.The H3K4me3 is associated with high transcription activity. However, LincRNA-EPS knockout does not affect the IFN-β induced H3K4me3 of ISGs in the activation state but not the rest state (Fig 5G and 5H). What are the reasons for the different effect？ Response: Thanks for your question and pointing out these unconvincing results. It is possible that too high concentration of IFN- treatment (500 U/mL) lead to the sutured induction of H3K4me3 interacting with ISG promoters, which caused almost no difference between lincRNA-EPS -/and WT groups in the activation state. We repeated this experiment by using optimized IFN- concentration or viral RNA mimics polyI:C. As shown in Fig 5G of the revised manuscript, the transcription activities of ISG were elevated in the lincRNA-EPS -/-iBMMs than the WT group, both in the resting and polyI:C-triggered activation states.

4.The authors claim that lincRNA-EPS regulates STAT1 activation and subsequently controls ISGs expression. However, the study only examined the effects of lincRNA-EPS on virus replication and tissue damage in vivo. Whether lincRNA-EPS could control ISGs expression in tissues (such as lung, spleen or liver) or serum?
Response: Thanks for your comments and suggestions. As suggested, we have repeated the VSV infection experiment in vivo, and measured the ISGs level as well as the viral load in tissues of liver and lung. Less viral load, lower IFN-, but higher ISG expression were observed in livers and lungs from the lincRNA-EPS -/mice than the WT mice (Figs 3I, J, EV4B and C of the revised manuscript), which indicate that lincRNA-EPS controls host antiviral immunity against VSV by regulating ISG expression in tissues. Fig 6H and 6I, lincRNA-EPS overexpression competitively binds virus RNA, which in turn inhibits PKR activation, but more evidence is needed to support this conclusion. Whether lincRNA-EPS knockout or knockdown could promote PKR binds RNA? Whether other dsRNA (such as poly(I: C)

2.Some description of the background of VSV-N and G is required.
Response: Thanks for your suggestion. We have added the background of VSV-N and G in the Results section (line 271-274). Fig 3G, the survival percentage of the lincRNA-EPS knockout group did not appear to be calculated from 100%.

If the authors conducted an RNAseq experiment of EPS-WT and EPS-KO after PKR down-regulation, it may help provide insights into the claimed PKR/EPS-dependent ISG signature -which should only be a portion of the ISGs see in EPS-WT. KO or down-regulation of such PKR-dependent ISGs in the context of EPS-KO cells with infection would be the only direct way to link EPS-PKR targets and antiviral effects.
Response: Thanks for your suggestion. We have conducted an RNA-seq experiment of resting WT and lincRNA-EPS -/-iBMMs after PKR down-regulation. However, we did not find specific genes regulated by hnRNPL-EPS axis or PKR-STAT1 axis.

Knockdown of PKR significantly downregulated numerous ISGs expression in both
WT and lincRNA-EPS -/-iBMMs. However, the fold changes of ISG expression between siNC and siPKR groups are not significant (Appendix Figure S1 of the revised manuscript). Therefore, we think that hnRNPL-EPS axis and PKR-STAT1 axis synergistically control ISGs expression but not work separately. More detail answer is in the 3 rd questions of Reviewer#1.
1st Mar 2022 1st Revision -Editorial Decision Dear Prof. Ma, Thank you for the submission of your revised manuscript. We have now received the enclosed reports from the referees that were asked to assess it. Referee 1 still has some minor suggestions that I would like you to incorporate before we can proceed with the official acceptance of your manuscript.
Referee 1 also assessed your reply to referee 2's concerns and noted that it is unclear whether technical or biological repeats were performed throughout the manuscript. This is a very important point. Technical repeats are performed in parallel in the context of one experiment, and biological repeats are independently performed experiments, on different days and ideally with different animals/cells/solutions/materials. Please make sure that all figure legends correctly state whether technical or biological repeats were performed. It is absolutely essential that the most important experiments in your study are performed at least 3 times independently, and the mean data of these independent experiments (plus statistical analyses) should be shown in the figures.
A few other editorial requests also need to be addressed: -Please correct the subheading to "Disclosure and Competing Interests Statement" and confirm that your statement is in line with our journal policy: https://www.embopress.org/competing-interests -All funding information also needs to be listed in our online manuscript system when you upload the final version of your manuscript. The online version is currently incomplete.
-If you like, you can change the APPENDIX figure into Figure EV6, as we do allow exceptionally 6 EV figures. Alternatively, you could also combine 2 EV figures to keep the maximum number of 5 EV figures.
-The EV figure legends need to be pasted to after the main figure legends in the main manuscript file. I attach to this email a related manuscript file with comments by our data editors. Please address all comments in the final manuscript.
I would like to suggest a few changes to the title and abstract. Do you agree with the following: LincRNA-EPS impairs host antiviral immunity by antagonizing viral RNA-PKR interaction LincRNA-EPS is an important regulator in inflammation. However, the role of lincRNA-EPS in the host response against viral infection is unexplored. Here, we show that lincRNA-EPS is downregulated in macrophages infected with different viruses including VSV, SeV, and HSV-1. Overexpression of lincRNA-EPS facilitates viral infection, while downregulation of lincRNA-EPS protects the host against viral infection in vitro and in vivo. lincRNA-EPS-/-macrophages show elevated expression of antiviral interferon-stimulated genes (ISGs), such as Mx1, Oas2, and Ifit2 at both basal and inducible levels. However, IFN-b, the key upstream inducer of these ISGs, is downregulated in lincRNA-EPS-/-macrophages compared with control cells. RNA pulldown and mass spectrometry results indicate that lincRNA-EPS binds to PKR and antagonizes the viral RNA-PKR interaction. PKR activates STAT1 and induces antiviral ISGs independent of IFN-I induction. LincRNA-EPS inhibits PKR-STAT1-ISGs signaling and thus facilitates viral infection. Our study outlines an alternative antiviral pathway, with downregulation of lincRNA-EPS promoting the induction of PKR-STAT1-dependent ISGs, and reveals a potential therapeutic target for viral infectious diseases.
EMBO press papers are accompanied online by A) a short (1-2 sentences) summary of the findings and their significance, B) 2-3 bullet points highlighting key results and C) a synopsis image that is exactly 550 pixels wide and 200-600 pixels high (the height is variable). You can either show a model or key data in the synopsis image. Please note that text needs to be readable at the final size. Please send us this information along with the revised manuscript.
I look forward to seeing a new revised version of your manuscript as soon as possible. Please use this link to submit your revision: https://embor.msubmit.net/cgi-bin/main.plex

Best regards, Esther
Esther Schnapp, PhD Senior Editor EMBO reports Referee #1: The authors have carefully addressed my comments in their rebuttal. However, the following points should be added to the manuscript itself. 1) The response to point 2 regarding the plausibility of EPS to bind to PKR given its low expression (about the comparison with CircPOLR2A) should be mentioned in the discussion. 2) Figure with siSTAT1 and its impact on MX1 and Oas2 should be added to the manuscript and discussed -as it directly links STAT1 and the observations. Referee #3: The authors have addressed all my concerns. I have no further comments.

Dear Dr. Esther Schnapp,
We are submitting our revised manuscript entitled "LincRNA-EPS impairs host antiviral immunity by antagonizing viral RNA interacting with PKR" (EMBOR-2021-53937V3) to EMBO Reports for consideration of publication.
We appreciate all the suggestions from you and referee 1. We have corrected the subheading to "Disclosure and Competing Interests Statement", listed all funding information in the online manuscript system, changed the APPENDIX figure into Figure EV6, pasted all the EV figure legends after the main figure legends in the main manuscript file. We have also revised the title and abstract as your great suggestions.
In addition, the experimental repeats have been checked and described in the figure legends.

The short summary of the findings and their significance:
The long non-coding RNA lincRNA-EPS binds to PKR in cytoplasm of macrophages, antagonizes viral RNA-PKR interaction, and thus impairs PKR-STAT1-dependent host antiviral immunity.

Bullet points:
• LincRNA-EPS is downregulated by host antiviral immunity • Knockout of lincRNA-EPS protects host against viral infection • LincRNA-EPS antagonizes viral RNA-PKR interaction • LincRNA-EPS impairs PKR-STAT1-dependent induction of antiviral ISGs A synopsis image with proper size and resolution has been submitted online.

Responses to the suggestions from Referee #1:
1) The response to point 2 regarding the plausibility of EPS to bind to PKR given its low expression (about the comparison with CircPOLR2A) should be mentioned in the discussion.

Response:
We have discussed the plausibility of cytoplasmic lincRNA-EPS binding to PKR by comparing the RNA copies per cell between lincRNA-EPS and CircPOLR2A (line 359-364 of the revised manuscript, yellow highlighted).

Response:
The results about siSTAT1 and its impact on MX1 and Oas2 have been shown in the Figure EV5G. We have also described and discussed these data (line 298-301 of the revised manuscript, yellow highlighted).
We think that we have addressed all the concerns raised by you and Referee 1.
Thank you very much for your consideration. We are looking forward to hearing from you.
With my best regards I am very pleased to accept your manuscript for publication in the next available issue of EMBO reports. Thank you for your contribution to our journal.
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